Therapy of the neoplastic diseases has largely involved the use of chemotherapeutic agents, radiation, and surgery. However, results with these measures, while beneficial in some tumors, has had only marginal effects in many patients and little or no effect in many others, while demonstrating unacceptable toxicity. Hence, there has been a quest for newer modalities to treat neoplastic diseases.
The Staphylococcal enterotoxins are a representative of a family of evolutionarily-related extracellular products of Staphylococcal aureus that belong to a well recognized group of proteins that have common physical and chemical and biologic properties known as superantigens. These proteins are which are the most powerful T cell mitogens known capable of activating 5 to 30% or the total T cell population compared to 0.01% for conventional antigens. Moreover, the enterotoxins evoke strong polyclonal T cell proliferation at concentrations 103-fold lower than conventional T cell mitogens. The most potent enterotoxin, Staphylococcal enterotoxin A (SEA), has been shown to stimulate DNA synthesis in human T cells at concentrations of as low as 10−13 to 10−16M. Enterotoxin-activated T cells produce a variety of cytokines, including IFNγ, IL-2 and TNFα. The Staphylococcal enterotoxins share common physicochemical properties such as heat stability, trypsin resistance, and solubility in water and salt solutions. Furthermore, the Staphylococcal enterotoxins have similar sedimentation coefficients, diffusion constants, partial specific volumes, isoelectric points, and extinction coefficients.
The enterotoxins are composed of a single polypeptide chain of about 30 kilodaltons (kD). SEA, SEB, SEC, SED, Staphylococcal toxic shock-associated toxin (TSST-1 also known as SEF), and the Streptococcal exotoxins share considerable nucleic acid and amino acid sequence homology. All staphylococcal enterotoxins have a characteristic disulfide loop near the middle of the molecule. SEA is a flat monomer consisting or 233 amino acid residues divided into two domains. Domain I comprises residues 31-116 and domain II of residues 117-233 together with the amino tail 1-30. In addition, the biologically active regions of the proteins are conserved and show a high degree of homology.
T cell recognition of SAgs, such as SEs, via the TCR Vβ region is independent of other TCR components and T cell diversity elements in a manner distinct from conventional antigens. Unlike conventional polypeptide antigens T cell activation by these molecules does not require antigen processing by an antigen presenting cell. They activate T cells by a biochemical signaling pathway distinct from conventional peptide antigens.
Single amino acid positions and regions important for SAg-TCR interactions have been defined. These residues are located in the vicinity of the shallow cavity formed between the two SE domains. (Lavoie P M et al., Immunol. Rev. 168: 257-269 (1999). SEB and the SEC bind only to the MHC class II β chain whereas SEA, SEE and SED, also interact with the MHC class II α chain in a zinc dependent manner. Substitution of amino acid residue Asn23 in SEB by Ala has demonstrated the importance of this position in SEB/TCR interactions. This particular residue is conserved among all of the SE's and may constitute a common anchor position for SE interaction with TCR Vβ structures. Amino acid residues in positions 60-64 of SEA contribute to the TCR interaction as do the Cys residues forming the intramolecular disulfide bridge (Kappler J et al., J. Exp. Med. 175 387-96 (1992)). For SEC2 and SEC3, the key points of interaction in the TCR Vβ region are located in the CDR1, CDR2 and HRV4 regions of the TCR Vβ3 chain (Deringer J R et al., Mol. Microbiol. 22: 523-534 (1996)). Hence, multiple and highly variable parts of the Vβ region contribute to the formation of the TCRs SE binding site. This distinctive binding mechanism of enterotoxins which bypasses the highly variable parts of the MHC class II and TCR molecules allows them to activate a high frequency of T cells resulting in massive lymphoproliferation, cytotoxic T cell generation and TH1 cytokines cytokine induction. Hence a given can activate up to 30% of resting T cells compared to 0.01% for conventional antigens.
Thus far, no single, linear consensus motif in the TCR vβ displaying a high affinity interaction with particular enterotoxins has been identified. A significant contribution of the TCRα chain in SE-TCR recognition is acknowledged (Smith et al., J. Immunol. 149: 887-896 (1992)). Unlike peptide binding in the groove between the MHCII alpha and beta chain, the SEs bypass the highly variable parts of the MHC class II and bind instead on the outer face of the groove. This distinctive binding to non-polymorphic regions of the MHCII endows them with their ability to activate such a high frequency of T cells and cause massive proliferation, cytokine induction and cytotoxic T cell generation. These properties are shared by several other proteins produced by various infectious agents. Together, these proteins form a well recognized family of molecules, SAgs, because of their aforementioned biological effects. Summary of the superantigen sequences that bind MHCII and vβ TCR regions is provided in Papageorgiou, A. C. et al. EMBO J. 18:9-21 (1999))
Wild type SEs and SE homologues and fusion proteins are known to induce anti-tumor effects. Administration of SEB produced antitumor effects against established tumors in two animal species, rabbits and mice, with tumors of five different histologic types: rabbit VX-2 carcinoma (Terman et al., U.S. Pat. No. 6,126,945; Terman, U.S. Pat. No. 6,340,461), murine CL 62 melanomas (Penna C. et al., Cancer Res. 54: 2738-2743 (1994)), murine A/20 lymphoma (Kalland T. Declaration in U.S. Ser. No. 07/689,799 (1992)), murine PRO4L fibrosarcoma (Newell et al., Proc Natl. Acad. Sci. 88: 1074-1079 (1991)) and human SW 620 colon carcinoma (Dohlsten et al., Eur. J. Immunol. 21: 1229-1233 (1991)). In these studies, parenterally-administered SEB induced objective anti-tumor effects at primary and metastatic sites. SEB was used ex vivo to stimulate a population of T cells pre-exposed to tumor, which, upon re-infusion into host animals with established pulmonary metastases, induced a substantial reduction of metastases. SEB activated T cell anti-tumor effect was specific for the immunizing tumor; the SEB stimulated T cells produced IFNγ which was thought to be an important mediator of the anti-tumor effect (Shu S et al., J. Immunol. 152: 1277-88 (1994)). Fusion polypeptides comprising SEA fused to a tumor specific monoclonal antibody (mAb), designated “SEA-mAb,” induced tumoricidal responses in the murine B16 melanoma model (Dohlsten M et al., Proc Natl Acad Sci 91:8945-9 (1994); Dohlsten M et al., Proc. Natl. Acad. Sci. 88:9287-91(1991). A summary of antitumor effects of superantigens is provided in Terman et al Clin Chest Med 27: 321-334 (2006).
The instant application provides a heretofore undescribed role for superantigens of boosting the tumoricidal response when fused recombinatly to a tumor associated antigen (TAA). Because superantigen and conventional antigens are aligned in geometrically different conformations on MHC II molecules required for activation of T cells such a fusion molecule would sterically interdict the binding of one of both its components to the MHCII receptor. Surprisingly, as shown herein in Example 1 and U.S. application Ser. No. 10/428,817 (of which the instant application is continuation in part) a nucleic acid construct encoding a superantigen fused to a weak TAA (papilloma viral epitope) abolished the outgrowth of squamous cell carcinoma in rabbits whereas nucleic acids encoding a superantigen or the viral epitope alone were ineffective. Further, parenteral delivery of tumor cells transduced with superantigen and a costimulatory molecule produced a tumoricidal response whereas mock transfected tumor cells similarly administered were ineffective (PCT/IS99/08399). Similarly, Bridle et al., (Mol Ther 18: 1430-1439 (2010) showed that immunization and boosting with a viral-nucleic acids encoding tumor associated antigen (TAA) construct resulted in a potent T cell response to the TAA and a tumoricidal effect that was not seen with the virus or tumor antigen alone. These results suggest that both superantigens and certain viruses (selected to induce altered self antigens) can combine with tumor associated epitopes to augment their immunogenicity in the host leading to an antitumor effect.
Thus, in the present invention nucleic acids encoding superantigens are fused to virus or viral genomic DNA (VASTA) capable of altering both tumor associated and self antigens upon transfection into tumor cells and normal cells of similar histologic type. This construct is used to transduce both tumor cells and normal cells of the same histologic type as the tumor. The cDNA from these cells is extracted from such cells and linked recombinantly to the original virus or a new VASTA and administered parenterally to the host. The final product consists of nucleic acids encoding a library of superantigen- and viral-altered normal and tumor cell associated antigens some of which are expressed as fusion proteins with the virus or superantigen. We hypothesize that the extracted cDNA containing nucleic acids encoding viral and/or SAg altered tumor associated self antigens is rendered highly immunogenic in the host by structural modification and/or fusion with the virus and the SAg. Systemic delivery of SAg-viral based nucleic acid libraries induce a broad repertoire of individually weak T cell responses against multiple TAAs resulting in a cumulatively powerful anti-tumor effect.
The present invention therefore exploits the ability of some viruses such as vesicular stomatitis virus to alter self antigens and render them immunogenic. cDNA from normal cells are used because cells transduced with virus express altered self antigens that when exposed to the host induce an immune response to antigen loss variants of tumor derived from these normal cells (Sanchez-Perez L et al., Caner Res 65: 2005-2017 (2009)). In addition, the present invention provides cDNA extracted from treatment resistant tumor cells because these cells express additional tumor epitopes such as cadherin and adhesion molecules not present on the original tumor or normal cells of the same histologic type or the primary tumor cells.
This unique therapeutic nucleic acid constructs derived from VASTA-SAg transfected tumor cells and normal cells of the same histologic type differs from viral constructs previously reported to treat cancer. Schlom et al discloses tumor cells transduced with nucleic acid construct comprising a virus-tumor associated antigen and costimulatory molecules that are administered directly into the tumor bearing host. In contrast to the instant invention, this nucleic construct does not contain a superantigen, is not extracted from transduced tumor cells and normal cells of the same histologic type, does not contain a library of superantigen/viral altered normal and tumor associated self antigens and uses an intact tumor cell as the therapeutic agent. Terman et al. (U.S. application Ser. No. 10/428,817) disclose administration of tumor cells transduced with nucleic acids encoding a superantigen and costimulatory molecules which differs from the instant invention in that it does not contain a virus and uses the transduced tumor cell as the therapeutic agent instead of cDNA extracted from VASTA-SAg transduced tumor cells and normal cells used in the instant application. Dow S W et al., (J Clin Invest 101:2406-43(1998) and Thamm D H et al., (Cancer Immunol Immunother 52:473-80 (2003)) disclose lipid complexed plasmid DNA encoding SEB and either granulocyte-macrophage colony-stimulating factor or IL-2 but this construct does not comprise tumor or normal cell-derived cDNA and is devoid of a virus selected to induce altered self antigens. To treat cancer, others have used fusion genes consisting of one or defined group of tumor/self antigens while some have used plasmid vectors that encode tumor antigens as in-frame chimeric fusions with other immune proteins (Englehorn et al., Mol. Ther. 16: 773-781 (2008)). None of these fusion agents however, use a superantigen in the fusion construct and none employ a complete cDNA library of modified self and tumor antigens extracted from tumor cells transduced with nucleic acids encoding SAg and VASTA.
In contrast to all of the above art, the cDNA extracted from tumor cells and normal cells and delivered to tumor bearing host contains not only nucleic acids encoding VASTA-SAg but also nucleic acids encoding self and tumor associated epitopes altered by VASTA-SAg. The immunogenicity of the altered TAAs and self epitopes are thereby augmented sufficiently to evoke a tumoricidal response. Unlike previous reports, the host is presented directly with the nucleic acids encoding a complete library of highly immunogenic self and TAAs together with the potent T cell adjuvant effects of SAg and a virus (VASTA) selected to induce altered self antigens in tumor cells and normal cells. To our knowledge, these nucleic acid therapeutic constructs encoding a library of altered self and tumor epitopes together with viral-superantigen-costimulatory molecules have not been previously employed to treat tumors.